Selenium is an essential micronutrient that exerts many important health benefits. The element is incorporated into selenoproteins as selenocysteine (Sec). The mammalian selenoproteins perform critical functions in anti-oxidant defense, thyroid hormone metabolism, male reproduction, and development. Sec is encoded by UGA, which is normally read as a stop codon. The recoding of UGA as Sec requires the Sec Insertion Sequence (SECIS) element in the selenoprotein mRNA. Although much progress has been made in understanding the mechanism of Sec incorporation, much less is known about the regulation of this pathway. In selenium deficiency, certain selenoproteins that are critical for health and development are expressed while other nonessential selenoproteins are lost. The central hypothesis of our proposal is that this complex hierarchy of selenoprotein expression is maintained by the interplay between multiple trans-acting factors that bind selectively to different SECIS elements. We discovered two new SECIS-binding proteins, nucleolin and eukaryotic initiation factor 4a3 (eIF4a3) that play opposing roles in selectively modulating selenoprotein synthesis. We showed that eIF4a3 links selenium status with differential selenoprotein expression. EIF4a3 is upregulated in selenium-deficient cells where it selectively inhibits the incorporation of Sec into two selenoproteins that perform nonessential functions. In preliminary studies, we identified new targets of eIF4a3 and developed a model of the eIF4a3: SECIS interaction. We also present evidence that hnRNP K binds selectively to a SECIS from a nonessential selenoprotein but not from an essential selenoprotein. In this project, we will elucidate the roles of eIF4a3 and hnRNP K in regulating the expression of the selenoproteome using a combination of in vitro assays, cell culture systems, and mouse models. The successful completion of this project will provide critical insight into how mammalian cells prioritize the utilization of selenium during selenium insufficiency, an important health problem in many parts of the world. By identifying these regulatory pathways, our studies will provide a strong foundation for developing more specific and targeted approaches for modulating selenoprotein expression in vivo.